Polyelectrolyte Coatings Can Control Charged Fluorocarbon

Subscriber access provided by University of Glasgow Library

Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Polyelectrolyte Coatings can Control Charged Fluorocarbon Nanodroplet Stability and their Interaction with Macrophage Cells Amanda L. Martin, Christa M Homenick, Yun Xiang, Elizabeth R. Gillies, and Naomi Matsuura Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b04051 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 19, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Polyelectrolyte Coatings can Control Charged Fluorocarbon Nanodroplet Stability and their Interaction with Macrophage Cells Amanda L. Martin†, Christa M. Homenick‡, Yun Xiang§, Elizabeth Gillies‡, and Naomi Matsuura§∥⊥* †

Physical Sciences, Sunnybrook Research Institute, Toronto, ON, Canada

‡

Department of Chemistry and Department of Chemical and Biochemical Engineering,

The University of Western Ontario, London, ON, Canada §

Department of Medical Imaging, University of Toronto, Toronto, ON, Canada

∥Department

of Materials Science and Engineering, University of Toronto, Toronto, ON,

Canada ⊥Institute

of Biomaterials and Biomedical Engineering, University of Toronto, Toronto,

ON, Canada *Corresponding Author: [email protected] The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

1 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 39

Keywords: fluorocarbon, nanodroplets, stability, polyelectrolyte, PEGylation, perfluorooctyl bromide

Table of contents


Page 3 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

ABSTRACT Fluorocarbon nanodroplets, ~100 to ~400 nm in diameter, are of immense interest in a variety of medical applications including the imaging and therapy of cancer and inflammatory diseases. However, fluorocarbon molecules are both hydrophobic and lipophobic, so it is challenging to synthesize fluorocarbon nanodroplets with the optimal stability and surface properties without the use of highly specialized surfactants. Here, we hypothesize that we can decouple the control of fluorocarbon nanodroplet size and stability from its surface properties. We use a simple, two-step procedure where standard, easily available anionic fluorosurfactants are used to first stabilize the fluorocarbon nanodroplets, followed by electrostatically attaching functionalized polyelectrolytes to the nanodroplet surfaces to independently control their surface properties. Herein, we demonstrate PEGylated polyelectrolyte coatings can effectively alter the fluorocarbon nanodroplet surface properties to reduce coalescence and its uptake into phagocytic cells in comparison to non-PEGylated polyelectrolyte coatings and uncoated nanodroplets, as measured by flow cytometry and fluorescence microscopy. In this study, perfluorooctylbromide (PFOB) was used as a representative fluorocarbon material, and PEGylated PFOB nanodroplets with diameters between 250 to 290 nm, depending on the PEG block length, were prepared. The PEGylated PFOB nanodroplets had superior size stability in comparison to uncoated and non-PEGylated polyelectrolyte nanodroplets in saline and within macrophage cells. Of significance, non-PEGylated nanodroplets were rapidly internalized by macrophage cells, whereas, PEGylated nanodroplets were predominantly co-localized on the cell membrane. This suggests that the PEGylatedpolyelectrolyte coating on the charged PFOB nanodroplets may afford adjustable


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 39

shielding from cells of the reticuloendothelial system. This report shows that using the same fluorosurfactant as a base layer, modularly assembled PFOB nanodroplets tailored for a variety of end applications can be created by selecting different polyelectrolyte coatings depending on their unique requirements for stability and interaction with phagocytic cells.


Page 5 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

INTRODUCTION Fluorocarbon nanodroplets, ~100 to ~400 nm in diameter, have unique properties that make them attractive in vivo materials for medical applications. In particular, there has been renewed interest in using emulsified fluorocarbon nanodroplets, with proven biocompatibility as demonstrated by their long history in patients as blood substitute agents,1 as therapeutic agents,2-8 radiosensitizers,9-13 and as multimodal contrast agents for computed tomography,14, 15 ultrasound imaging,7, 16-21 and magnetic resonance imaging (MRI)22-27 for applications including tumor imaging, inflammation imaging, and cell tracking. Due to the high strength of the carbon-fluorine bond, fluorocarbon molecules have high chemical and oxidative stability and are both hydrophobic and lipophobic, repelling both water and lipids.28 Although these properties lead to their low reactivity with biological compounds and good in vivo clearance characteristics, they also make it challenging to emulsify fluorocarbons into stable, size-controlled nanodroplets using standard, biocompatible surfactants required for in vivo applications.7, 26, 28 To-date, substantial research has been dedicated to formulating droplets with small diameters and with suitable stability in vivo, for example using lipids,29-31 polymers,32 proteins,33 and hydrocarbon surfactants34 as emulsifiers. Since these emulsifiers are amphiphilic, they are not miscible with fluorocarbons. Due to this low affinity between these emulsifiers and fluorocarbons, this combination results in relatively low stabilities and wide size distributions of nanodroplets after emulsification. Alternatively, fluorocarbon droplets with cross-linked polymer shells4, 18, 24 have good size stability but suffer from poor clearance, and these stiff solid shells preclude their use for imaging and therapy 5 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 39

applications that require a soft shell (e.g., ultrasound or photoacoustic contrast imaging4, 31, 35-37

and triggered drug delivery7).

On the other hand, commonly available fluorinated surfactants (i.e., fluorosurfactants) can stabilize fluorocarbon nanodroplets more effectively than hydrocarbon-based surfactants.33, 38 Furthermore, many specialized fluorocarbon droplet systems, for example, monodisperse droplets formed through microfluidics39-41 or extrusion,20 rely on fluorosurfactant stabilization. However, it has been shown that fluorosurfactant-stabilized fluorocarbon nanodroplets must be sterically repulsive for size stability in vivo, as electrostatically-stabilized fluorocarbon nanodroplets result in rapid nanodroplet coalescence at short inter-droplet distances in biological media.35, 42 Furthermore, the strong negative charge on the nanodroplet surface due to anionic fluorosurfactant stabilization can also enhance its uptake into cells.14, 35, 43, 44 To prevent rapid macrophage clearance and to increase the nanodroplets’ in vivo stability, new and specialized PEGylated fluorosurfactants19, 45, 46 or copolymers containing a fluorocarbon block are required.47 In addition, the heavy dependence of the surfactant on the nanodroplet size, stability and activation of the complement system means that all fluorocarbon nanodroplet formulations must be individually optimized to meet the widely varying requirements for each particular disease application. Here we explore the feasibility of decoupling the surfactant used to stabilize the fluorocarbon nanodroplets from the development and optimization of biomolecules used to control their surface properties. Specifically, we hypothesize that if fluorocarbon nanodroplets can first be stabilized using a negatively charged fluorosurfactant, then its surface properties can be further modularly adjusted according to the end application 6 ACS Paragon Plus Environment

Page 7 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

through the electrostatic deposition of a positively charged polyelectrolyte layer onto the nanodroplet surface. Fluorosurfactants tend to enhance fluorocarbon nanodroplet stability in comparison to common amphiphilic hydrocarbon-based surfactants due to the improved wetting of the fluorosurfactant around the fluorocarbon core arising from the fluorous interactions between the its fluorinated chains and the fluorocarbon.19, 45 The direct electrostatic interaction between the fluorosurfactant and the particular polyelectrolyte, for example, a PEGylated polyelectrolyte, as a method of attachment avoids the potentially complicated and time-consuming chemical conjugation methods of fluorosurfactant PEGylation or even totally new fluorosurfactant syntheses. As the same fluorosurfactant is used as the base layer, modularly assembled fluorocarbon nanodroplets can be readily designed for different disease targets and applications using the wide range of bioconjugated and functionalizable polyelectrolytes available today without having to develop new application-specific surfactants from scratch. In this study, we evaluate whether different polyelectrolyte coatings can modulate the ability of fluorosurfactant-stabilized fluorocarbon nanodroplets to coalesce and be taken up by cells, and assess the potential of using polyelectrolyte coatings to control the nanodroplet size and surface-dependent performance to design for different end applications in vivo. Here one type of commonly used fluorocarbon, perfluorooctyl bromide (PFOB, C8F17Br), was applied as a model fluorocarbon. The incorporation of bromide in the PFOB molecule, unlike in other fluorocarbons, means that PFOB is slightly more lipophilic than pure fluorocarbons, making the fluorosurfactant stabilized PFOB nanodroplets slightly less size stable than fluorosurfactant stabilized perfluorocarbon nanodroplets. The metastable nature of fluorosurfactant-stabilized PFOB


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 39

nanodroplets, in addition to the high PFOB boiling point of 142°C at 1 atm, makes these droplets ideal for this study to reveal the efficacy of stabilization derived from the deposition of polyelectrolytes.28 Specifically, an anionic fluorosurfactant (FSP)-stabilized PFOB nanodroplets were formulated and later separately coated by cationic polyelectrolytes with different poly (ethylene glycol) (PEG) lengths (i.e., two PEG conjugated poly-L-lysines (PLLs), where 25% of the lysine functional groups were coupled to either 2000 or 5000 g/mol PEG acid) and their stability in vitro was assessed in comparison to uncoated, anionic, FSP-stabilized PFOB nanodroplets and cationic, nonPEGylated PLL-coated PFOB nanodroplets. The effect of different PFOB nanodroplet polyelectrolytes on macrophage uptake was assessed by evaluating their interaction with macrophage cells in vitro in comparison to control nanodroplets using flow cytometry and fluorescence microscopy. EXPERIMENTAL SECTION Materials Perfluorooctylbromide (PFOB, C8F17Br, b.p. 142°C at 1 atm) was purchased from SynQuest Laboratories (Alachua, FL, USA). Poly (ethylene glycol) monomethyl ether monoacetic acid ether (PEG-COOH) with a molecular weight of 2000 g/mol or 5000 g/mol was purchased from JenKem Technology (Plano, Texas, USA). Solvents were purchased from Caledon Laboratory (Georgetown, ON, Canada). Zonyl FSP fluorosurfactant (CAS# 67479-86-1) and all other chemicals were purchased from SigmaAldrich (St. Louis, MO, USA). All chemicals and solvents were used as received unless otherwise indicated. Dry dimethylformamide (DMF) was obtained from an Innovative


Page 9 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Technology (Newburyport, MA, USA) solvent purification system based on aluminum oxide columns. Deionized water (Millipore, Burlington, MA, USA, Milli-Q grade, 18.2 M) was used in all experiments. Molecule characterization 1

H NMR spectra were obtained in CDCl3, and D2O using Varian Inova 600 MHz and

Mercury VX 400 MHz spectrometers (Agilent Technologies, Santa Clara, CA, USA). NMR chemical shifts are reported in ppm and are calibrated against residual solvent signals of CDCl3 (δ 7.26) or D2O (δ 4.80). Size exclusion chromatography (SEC) in DMF was carried out at a flow rate of 1 mL/min in DMF containing 10 mM LiBr and 1% (v/v) NEt3 at 85 C using a Waters 2695 separations module equipped with a Waters 2414 refractive index detector (Waters Limited, Mississauga, ON, Canada), two PLgel 5 m mixed-D (300 mm  7.5 mm) columns and the corresponding guard column (Agilent) connected in series. Aqueous SEC was performed at ambient temperature in pH 7.4, 0.1 M phosphate buffer containing 10 mM NaN3 using a Waters 515 HPLC pump equipped with a Wyatt Optilab T-rEX refractometer, two PL aquagel-OH Mixed M (300 mm x 7.5 mm) columns and the corresponding guard column (Agilent). For both DMF and aqueous SEC the Mn and Đ were determined relative to PEG standards (Agilent). Synthesis of PLLHCl (2) Carboxybenzyl (CBz)-Protected PLL (1) was synthesized as previously reported and the product had an Mn of 7270 and Đ = 1.16 based on SEC in DMF relative to PEG standards.48 A round bottom flask equipped with a Teflon stir bar was charged with CBzprotected PLL (1) (0.43 g, 5.7 mmol of amine, 1 eq.), and trifluoroacetic acid (TFA) (10 9 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 39

mL, 0.13 mol, 23 eq.). The round bottom flask was fitted with a condenser and placed in a pre-heated oil bath to 60 °C. The reaction was stirred overnight. The resulting reaction mixture was cooled to room temperature, diluted with 6 mL of HCl/Ether (1:1), and subsequently precipitated in acetone. The resulting pale yellow powder was isolated by vacuum filtration. Yield: 0.31 g, pale yellow powder (79 %). 1H NMR (600 MHz, D2O): δ 1.31 – 1.50 (br, 2H), 1.61 – 1.83 (br m, 4H), 2.91 – 3.03 (br m, 2H), 4.25 – 4.32 (br m, 1H). Synthesis of PLL-g-2K-PEG (4a) PLLHCl (2) (100 mg, 0.59 mmol of amine, 1.0 eq.), PEG-COOH (3a) (0.29 g, 140 μmol, 0.25 eq. relative to amine) and 1.5 mL of distilled water were combined in a vial. The pH of the reaction mixture was adjusted to pH = 5.5 utilizing 1N HCl and 1N NaOH. In a separate vial, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDCHCl) (55 mg, 0.29 mmol, 0.5 eq. relative to amine) and N-hydroxysuccinimide (NHS) (42 mg, 0.37 mmol, 0.6 eq. relative to amine) were dissolved in 1.5 mL of water. The two reaction mixtures were combined and stirred overnight. The resulting reaction mixture was dialyzed against water using a regenerated cellulose membrane with a molecular weight cut-off of 25000 g/mol for 24 hours. The remaining solution was lyophilized to yield a white powder. Yield: 0.285 g, white powder (71 %). 1H NMR (600 MHz, D2O): δ 1.25 – 1.55 (br, 2H), 1.55 – 1.91 (br, 3.7H), 2.89 – 3.10 (br, 1.9H), 3.36 (s, 1.1), 3.51 – 3.84 (br m, 62H), 3.92 (s, 0.6), 4.24 – 4.37 (br, 0.9H). SEC (aqueous): Mn = 26050, Đ = 1.04. Synthesis of PLL-g-5K-PEG (4b)


Page 11 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

PLLHCl (29 mg, 0.18 mmol of amine, 1.0 eq.), PEG-COOH (3b) (0.21 g, 43 μmol, 0.25 eq. relative to amine) and 1.5 mL of distilled water were combined in a vial. The pH of the reaction mixture was adjusted to pH = 5.5 utilizing 1N HCl and 1N NaOH. In a separate vial, EDCHCl (19 mg, 98 μmol, 0.5 eq. relative to amine) and NHS (14 mg, 130 μmol, 0.7 eq. relative to amine) were dissolved in 1.5 mL of water. The two reaction mixtures were combined and stirred overnight. The resulting reaction mixture was dialyzed against water with a molecular weight cut-off of 50000 g/mol for 24 hours. The remaining solution was lyophilized to yield a white powder. Yield: 0.186 g, white powder (74 %). 1H NMR (600 MHz, D2O): δ 1.27 – 1.55 (br, 2H), 1.55 – 1.90 (br, 3.2H), 2.88 – 3.10 (br, 1.7H), 3.36 (s, 1.1), 3.51 – 3.85 (br m, 168H), 3.92 (s, 0.7H), 4.22 – 4.39 (br, 0.6H). SEC (aqueous): Mn = 50300, Đ = 1.04. Preparation of fluorosurfactant stabilized PFOB nanodroplets A fluorescently-labeled PFOB solution was first prepared using a previously published and validated procedure.14, 43, 49 CdSe/ZnS core/shell quantum dots were used as the fluorescent tag, as they have previously been shown to have no measurable impact on the fluorocarbon nanodroplet stability and their interaction with target cells.14, 35, 43 Negatively-charged, PFOB nanodroplets stabilized with anionic fluorosurfactant (FSP) was prepared by sonication.14 Briefly, 15 μL of FSP was dissolved in 2 mL of deionized water by sonication in a water bath. To this mixture, the PFOB solution (70 μL) was added, the mixture was suspended in a cold-water bath and sonicated with a Branson Digital S450D Sonifier (Emerson Canada, Markham, ON, Canada) for 5 minutes of exposure at 10 % amplitude (2.5 W), using a 1 second on 1 second off pulse with a 1/8” titanium microtip. Samples were centrifuged using an Eppendorf 5430 Centrifuge 11 ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 39

(Eppendorf Canada, Mississauga, ON, Canada) at 3000 rpm for 20 minutes. The supernatant with excessive fluorosurfactant was removed, and the remaining pellet was redispersed in deionized water (2 mL) with gentle shaking. Hydrodynamic diameters and zeta potentials were measured using a Malvern Zetasizer Nano-ZS 3000HS (Malvern Instruments, Worcestershire, UK) dynamic light scattering (DLS) instrument. Preparation of polyelectrolyte coated PFOB nanodroplets Each polyelectrolyte (PLL or PEGylated PLLs) was dissolved in 0.01M NaCl solution to a concentration of 0.5 mg/mL (PLL) and 1 mg/mL (PLL-g-N-PEG, N = 2K or 5K). PFOB nanodroplets (0.5 mL) were added dropwise into a stirring solution of the polyelectrolyte of interest (2 mL) and stirred for 10 minutes to ensure the full coverage. The coating progress was monitored by measuring changes in zeta potential and size of the nanodroplets by DLS. Each coated PFOB nanodroplet sample was then centrifuged to remove excess and non-adhered polyelectrolyte in the supernatant at 3000 rpm for 10 minutes. Nanodroplets were redispersed in deionized water (0.5 mL) with gentle shaking. Redispersed coated and uncoated PFOB nanodroplets were then further centrifuged at low rpm (1000 rpm, 5 minutes) to remove any micron-scale aggregates in the pellet, due to the partial coating of some nanodroplets. Fluorescence emission spectra of the quantum dot labeled nanodroplets were acquired on a Horiba Jobin Yvon FluoroMax-4 Spectrofluorometer (Horiba Scientific, Edison, NJ, USA). Samples were excited at λex = 400 nm using a slit width of 1 nm, and the emission spectrum from 500 nm to 700 nm was scanned using a slit width of 1 nm at 1 nm increments. The fluorescence peak of the quantum dots was determined to be at λem = 624


Page 13 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

nm. All the nanodroplet samples were measured and normalized with dilution such that all nanodroplet samples had the same fluorescence intensity. Since the quantum dot to PFOB ratio within each batch of PFOB nanodroplets was the same, the normalized nanodroplet samples with different coatings were determined to have similar PFOB concentrations. Stability assessment in solution To measure the stability in water, PFOB nanodroplets were kept in centrifuge tubes at room temperature for up to 1 week and the size and zeta potential re-measured over time with DLS. For stability in saline, aliquots of 0.2 mL nanodroplets (of each type) were added to 2 mL saline in a 6-well plate, kept at room temperature, and imaged at 40x magnification with a high-speed camera (CoolSNAP HQ2, Photometrics, Tuscon, AZ, USA). Nanodroplets were observed for 2 hours after addition and reassessed at 24 hours. Droplets that could be observed using optical microscopy were analyzed using Image-Pro Plus software (Media Cybernetics, Bethesda, MD, USA) and ImageJ software (NIH, http://rsb.info.nih.gov/ij/) to generate size histograms.50 Each histogram was based on multiple images for each droplet variant, such that more than 3000 particles were measured for each histogram. Control images containing no nanodroplets were also analyzed to determine the background noise to ascertain the lower droplet size limit that could be measured. Here, particles  0.7 m were not included in the histogram analysis. After 24 hours, the nanodroplets were collected for size and zeta potential measurement with DLS. Interaction with macrophage cells


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 39

Cellular uptake and changes in the size of PFOB nanodroplets were evaluated in vitro using murine alveolar macrophage cells (RAW264.7, ATCC, Manassas, VA, USA). During cell growth and incubation with nanodroplets, the cells were maintained at 37 °C and 5 % CO2 atmosphere in Dulbecco’s modified essential medium (DMEM) with 4.5 g/L glucose, L-glutamine, and sodium pyruvate supplemented with 10 % heat-inactivated fetal bovine serum (Wisent Inc., St-Jean-Baptiste, QC, Canada). Cells were seeded at 8 × 105 cells/well in 6-well plates and allowed to adhere and grow until 80 % confluency was reached, both with and without glass slides for fluorescence microscopy and flow cytometry respectively. Cells were incubated with PFOB nanodroplets for 2 hours at 40fold dilution to minimize changes in nanodroplet size outside of cells.35 Flow cytometry was applied to determine the interaction between PFOB nanodroplets and cells. After incubation and washing, cells were detached by scraping into tubes containing 4 % paraformaldehyde (PFA), where they were fixed for 30 minutes. Tubes were then centrifuged at 3600 rpm and 4 ˚C for 5 minutes. The supernatant was removed, and cells were redispersed in 1 mL sterile PBS with repeated pipetting. PFOB nanodroplet uptake was determined on a BD FACSCalibur flow cytometer (BD Biosciences, Mississauga, ON, Canada) with filters optimized for the emission spectra of the quantum dot fluorescent tags, with the same voltage setting for each sample set. The autofluorescence from the cell was acquired from the wells not incubated with the nanodroplets. Six independent experiments were conducted to assess PFOB nanodroplet uptake into cells. Flow cytometry data were gated to exclude small cell debris and free PFOB nanodroplets, and analyzed using FloJo software (TreeStar, Ashland, OR, USA). To test the statistical significance of the change in uptake of the labeled nanodroplets, t-


Page 15 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

tests were performed between the desired sample sets. Values were considered significant for p

Polyelectrolyte Coatings Can Control Charged Fluorocarbon

Recommend Documents